Summary The composition and physiology of the microbial community (microbiota) in the human colon has been linked to a number of diseases. Mechanistic details for most of these interactions are still badly needed. The shared focus of the four investigators assembled to conduct the proposed project is to understand how gut microbes interact with and metabolize complex carbohydrates?especially the glycans attached to secreted host mucus. Mucus is the first barrier that separates intestinal bacteria from host tissue and is a complex mixture of secreted mucin glycoprotein and other molecules. Some bacteria have evolved to forage on mucus as a source of nutrients. We have previously shown that this mucus foraging activity increases when exogenous dietary fiber polysaccharides are absent. Using a gnotobiotic model of fully sequenced human gut bacteria, we have shown that during fiber deficiency the gut microbiota resorts to degrading mucus for nutrients, leading to erosion of its integrity. In wild-type mice, a reduced mucus barrier increases epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. More strikingly, when this same synthetic microbiota is assembled in mice deficient in interleukin 10, a cytokine for which loss of function is associated with human pediatric inflammatory bowel disease (IBD), animals develop lethal inflammation in the absence of pathogen, but only on a low fiber diet. Our work has therefore revealed functional connections between mucus integrity, diet and gut microbes in precipitating IBD. The complete deconstruction of mucin glycoproteins requires a consortium of enzymes: peptidases to hydrolyze the protein backbone and sulfatases and glycoside hydrolases that recognize sulfated or unsulfated oligo- and monosaccharides within discrete glycosidic linkage contexts. Our central hypothesis is that mucin is degraded in a series of sequential steps by individual activities in this enzyme consortium and that essential catalytic steps exist, which may be contributed by different species that work synergistically to degrade mucus. We will test this hypothesis by first defining the sequential action, positional specificity and key structural facets of bacterial enzymes required for degradation of gastrointestinal mucins. We will use sequential and combinatorial treatments of various forms of mucin with pure recombinant enzymes, which we have already identified in the members of our synthetic microbiota. In parallel, we will measure the requirement for individual, discrete mucus-degrading steps within genetically- manipulable model species using in vitro and mouse in vivo models as readouts. The research team is composed of four leaders in the disciplines of gut bacterial physiology and molecular biology, structural biology and enzymology, mucin biology and glycoanalytics, all with a shared interest in the mechanisms of mucus degradation and the consequences for human disease. Successful completion of these experiments will define a precise series of mechanistic steps for bacterial mucin degradation and could lead to therapies to limit these events in diseases like IBD.